Whether apoptotic stimuli arise from the nucleus, cell membrane surface, or the mitochondria, ultimately, the stimuli converge on a process of activation of a family of cysteine proteases known as the caspases (cysteine aspartases). Activation of members of the caspase family have been shown to be necessary for programmed cell death in a number of biological systems and evidence strongly points to caspase-3 as standing at the center of the execution pathway of the cell death program. While existing in a non-active pro-enzyme form in the cytosol of resting cells, caspase-3 is one key "effector" protease when activated. Thus, to monitor the final commitment of tumor cells to death pathways, the need exists to directly quantify the enzymatic activity of caspase-3 in vivo. To meet this challenge, we have designed and synthesized prototype of an entirely new class of peptide based imaging agents that can permeate the cell membrane. Incorporating permeation motifs of viral proteins, these prototypic membrane permeant peptide conjugates are capable of translocating across the cell membrane to the cytosolic compartment. Engineered with substrate recognition sequences and appropriate peptide-based motifs for chelating radionuclides such as technetium-99m (Tc-99m), these agents will enable imaging of caspase-3 activity in vivo. Enzyme-dependent signal amplification should result in high quality enzyme-specific molecular images. In this way, focal retention of radioactivity, a "hot spot", will be generated on tumor images where there exists enzymatically active caspase-3. We propose to define the structural determinants of the viral protein cell membrane permeation sequences for use in peptide imaging conjugates, use a degenerate peptide combinatorial library to determine the preferred recognition sequences of the peptide imaging substrates, define the structural determinants and charge of the chelation moiety of our peptide conjugates that will confer favorable cellular retention properties to the imaging fragments, and perform pre-clinical evaluation of Tc-99m labeled peptide chelation conjugates in order to study the time course of activation of caspase-dependent apoptosis in tumor models grown in nude mice. Studies with these novel imaging tools will assist interrogation of the efficacy of new therapies, including gene therapy and new molecular targeted chemotherapy in cancer.